The bacteria genus Streptococcus includes some of the most important human pathogens, causing a wide range of different disease, and inflicting significant morbidity and mortality throughout the world. Our long term goal is to reach a thorough understanding of the molecular specifics correlated with adaptive differences within and between the pathogenic taxa of the genus Streptococcus. Our central hypothesis is that many loci, sites within loci, and noncoding functional elements, identified as being under lineage specific, or positive selection pressure, in our evolutionary analyses, will be key loci involved in the colonization, persistence, survival, and propensity to cause disease in these pathogenic streptococci. The specific aims of this proposal can be summarized as follows: 1. Generate and annotate genome sequence data for 9 species of Streptococcus within the Pyogenes, and Mutans groups, chosen such that they will yield various sister group comparisons involving important human pathogens, and construct a Streptococcus Genome Browser for presentation of data and associated comparative genomic analyses. 2. Assess the role of positive selection and lateral gene transfer in the diversification of the genomes of the species within the different Streptococcus groups. 3. Collect comparative sequence data of species specific, putative virulence, and core genes, across strains of the same species, for the purpose of analyzing selection pressure and demographic history, employing new population genetic based methods developed by us, as well as additional methods developed as part of this project. 4. Employing new methods developed by us, as well as additional methods developed as part of this project, conduct a comprehensive survey of noncoding functional elements in the Streptococcus Pyogenes, Mutans, and Mitis groups. 5. Based on the results arising from the selection analyses, and identification of noncoding functional elements, create gene knockout and site specific mutants for S. mutans, and assay the phenotypic effects. The fundamental data on comparative genomics and molecular adaptation, arising from this project, will serve as a framework for the development of novel therapeutic and preventative strategies for pathogenic species of Streptococcus. The results from these evolutionary analyses will serve as a guide for future follow-up, cause- effect experimentation, and the Streptococcus Genome Browser will serve as a valuable resource to the scientific community by displaying results of in vitro and in vivo studies of associations between sequence and function. Project Narrative: This project will yield a great deal of fundamental information regarding the molecular details of how Streptococcus pathogens are adapted to their human hosts. This information will in turn be a valuable asset to the development of novel therapeutic and preventative strategies for these pathogenic bacteria.